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  • Thus DGK active sites remain ill defined

    2021-06-19

    Thus, DGK-active sites remain ill-defined and, combined with the lack of crystal structures for mammalian DGKs, have limited our understanding of substrate and inhibitor binding. As a result, current DGK inhibitors consist of compounds with poor specificity within the DGK superfamily (de Chaffoy de Courcelles et al., 1985, de Chaffoy de Courcelles et al., 1989) or lack selectivity measurements against other lipid and protein kinases (Boroda et al., 2017, Liu et al., 2016, Purow, 2015). Thus, methods that provide information on small-molecule binding mode and selectivity are needed to guide development of isoform-selective DGK inhibitors. Selective DGK inhibitors are needed to study isoforms where knockout mice viability is an issue (Crotty et al., 2006) and to help realize the translational potential of targeting specific forms, e.g., DGKα, for anticancer (Dominguez et al., 2013) and immunotherapy applications (Prinz et al., 2012). Here, we use ATP acyl phosphate activity-based probes (Patricelli et al., 2007, Patricelli et al., 2011) and quantitative mass spectrometry to discover ATP and inhibitor AR 231453 of representative members of all five principal DGK subtypes. Our findings define, for the first time, the ATP binding motif of DGKs that is distinct from protein kinases and identifies the DAGKa subdomain as a novel region mediating ATP binding. We discovered a fragment of the DGKα inhibitor ritanserin that shows conservation of binding mode and enhanced selectivity against protein kinases, supporting the concept that the atypical C1 and accessory region of the catalytic domain (DAGKa) are key ligand binding sites for developing DGKα-selective inhibitors. Our studies demonstrate the utility of chemical proteomics to map ligand binding sites for fragment-based discovery of lipid kinase inhibitors.
    Results
    Discussion We used ATP acyl phosphates and quantitative LC-MS to map ligand binding regions corresponding to the active site of mammalian DGKs. We defined, for the first time, the location of the ATP binding site of representative isoforms from all five principal DGK subtypes (Figure 5). Inspection of the DGK ATP binding sites reveals several important features that are unique to this lipid kinase family. First, we identified ATP-sensitive, probe-modified peptides from both DAGKc and DAGKa subdomains, supporting interactions between these regions within the catalytic domain to constitute a potential ATP binding cleft. Crystal structures of soluble bacterial lipid kinases with homology to mammalian DGKs have also been found with active sites located in an interdomain cleft (Bakali et al., 2007). Our finding that the DAGKa region is involved in substrate binding was important for assigning a catalytic role to this domain, and helps explain previous reports that C-terminal truncations impair DGK enzymatic activity (Los et al., 2004). Second, conserved sequences corresponding to ATP binding sites of DGKs (Figures 5B and 5C) are not homologous with glycine-rich loops mediating ATP binding of protein kinases (Hanks et al., 1988, Hemmer et al., 1997). Our data provide the first experimental evidence in support of a unique DGK ATP binding motif that was postulated over 20 years ago (Schaap et al., 1994). Finally, it is tempting to speculate that detection of a single ATP binding site (as opposed to two sites in other DGKs) for DGKκ and DGKε is a reflection of functional differences in substrate binding of DGK subtypes (Figure 5A). In support of this hypothesis, DGKκ, along with other type 2 members, contain an unusual peptide motif that physically separates the DAGKc and DAGKa subdomains (Imai et al., 2005). DGKε, the sole type 3 member, is the only subtype that lacks regulatory domains and shows acyl chain preference in DAG substrate assays in vitro (Tang et al., 1996). We should note that DGKκ and DGKε showed lower recombinant protein expression compared with other isoforms (Figure S1), and so we cannot rule out the possibility of detection limits using our LC-MS approach. Future studies will be required to evaluate how these distinctions in active sites influence substrate (DAG) specificity and function across DGK subtypes.